Total internal reflection occurs when highly inclined light impinges upon a refractive index boundary (with first medium index n1 and second medium index n2, with n1>n2). According to Snell's Law, n1 sin(theta1)=n2 sin(theta2), where theta1 is the angle of incoming light (measured from the surface normal) and theta2 is the corresponding outgoing angle for the refracted light. For theta2=pi/2, theta1=arc sin(n2/n1) is the “critical angle”. Any light ray at or exceeding the critical angle is “totally internally reflected”, and no light propagates past the interface into the far field on the lower index side of the boundary. Nevertheless, an evanescent wave exists at the interface of the boundary and can excite fluorescent molecules within lambda (the wavelength of excitation) distance from the boundary, on the n2 side. This evanescent wave is used in TIRF to generate very high contrast, high signal-to-noise ratio images of fluorescently-labeled samples, such as the cell membrane, at or near the coverslip boundary.
A convenient method of setting up TIRF conditions is to use a high numerical aperture objective (typically 1.4 NA or higher, so that aqueous samples with n2˜1.33, the refractive index of a cell may be imaged) and ensure that only marginal rays pass through the back focal plane of the objective lens. Such “objective side TIRF” has been very successful in cell biology applications, where it has been used for decades.
Structured illumination microscopy (SIM) is a method that uses sharply patterned light and post-processing of images to enhance image resolution (in its linear form, doubling resolution). In traditional SIM, a series of images are acquired with a camera and computationally processed to improve resolution. This implementation of SIM has also been combined with TIRF, but the implementation still requires 9 raw images relative to normal TIRF microscopy, thereby slowing acquisition 9-fold relative to conventional, diffraction-limited imaging. As such, there is a need for a method that combines SIM with TIRF conditions that does not result in a loss of speed relative to conventional TIRF microscopy.
It is with these observations in mind, among others, that various aspects of the present disclosure were conceived and developed.
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